313 IN VITRO MATURATION AND FERTILIZATION OF OOCYTES COLLECTED FROM UNSTIMULATED MACACA NEMISTRINA OVARIES

Abstract

Reports describing the IVM of Macaca nemestrina (Mn) oocytes are limited (Cranfield MR et al. 1989 Zoo. Biol. (Supp. 1), 33). The use of gonadotrophins (Gnt) for IVM of non-human primate (NHP) oocytes is common but the concentrations used are often high (8–40 IU mL⁻¹) and the species of origin and biological activity of Gnt varies (Schramm RD and Paprocki AM, 2000 Hum. Reprod. 15, 2411). We have compared two different IVM systems with human Gnt on maturation and fertilization of oocytes collected from unstimulated Mn ovaries (n = 6–10 animals). Oocytes were subjected to IVM in modified (minus PVA and pantothenic acid, plus 20 amino acids) HECM −10 + 15% FCS (Zheng P et al., 2001 Mol. Reprod. Dev. 58, 348) for a) 36 h in the presence (mHECM +36, n = 322) or absence (mHECM −36, n = 99) of FSH and LH applied sequentially (FSH 1 IU mL⁻¹ 0–24 h; 10 IU mL⁻¹ FSH and LH 24–36 h) or b) 24 h in the presence (mHECM +24, n = 119) or absence (mHECM −24, n = 56) of static concentrations of Gnt (FSH and LH 1 IU mL⁻¹ 0–24 h; no Gnt 24–30 h). Oocytes exhibiting first polar body extrusion at 36 and 30 h were recorded as mature (MII) and subjected to IVF in HTF + BSA (3 mg mL⁻¹) with Mn sperm pretreated with 1.0 mM caffeine and 0.1 mM dbcAMP. Fertilized oocytes (pronuclei and/or 2nd polar body extrusion) were cultured in sequential culture medium for 48 h, assessed for cleavage and either fixed or frozen. Proportional data (mature/total, fertilized/mature or cleaved/fertilized) were compared by chi-square analysis and are reported as percentages. Oocytes cultured in mHECM +36 and mHECM −36 exhibited similar rates of GVBD (58.7% v. 53.5%) but the percentage of MII oocytes was significantly higher (P = 0.0244) in mHECM +36 (41.3%) v. mHECM −36 (28.3%). Fertilization rates were comparable between mHECM +36 (61.5%) and mHECM −36 (60.9%), whereas cleavage rates were significantly higher (P = 0.0004) in mHECM +36 (74.6%) v. HECM −36 (21.4%). Oocytes cultured in mHECM +24 and mHECM −24 exhibited similar rates of GVBD (76.5% v. 62.5%) but the proportion of MII oocytes was significantly higher (P = 0.0159) in mHECM +24 (55.5%) v. mHECM −24 (35.7%). Fertilization and cleavage rates were comparable between mHECM +24 (58.8% v. 63.3%) and mHECM −24 (50.0% v. 42.8%). A comparison between mHECM +36 and mHECM +24 indicated a significantly lower (P = 0.0005) percentage of GV oocytes and a significantly higher (P = 0.0096) percentage of MII oocytes in mHECM +24 (23.5% v. 55.5%) compared to mHECM +36 (41.3% v. 41.3%). Fertilization and cleavage rates were not significantly different between mHECM +36 and mHECM +24. Oocyte maturation and fertilization and embryo cleavage were not different for mHECM −36 and mHECM −24 (P = 0.3138–0.8202). Mn oocytes exhibit high rates of Gnt-independent GVBD (52.5%–53.5%) and maturation (28.3%–35.7%) in vitro, and maturation rates were improved in Gnt supplemented maturation medium. However, reduced exposure to lower concentrations of FSH and increased exposure to lower concentrations of LH was associated with higher rates of oocyte maturation in vitro. The use of lower concentrations of FSH and LH for reduced periods may improve IVM of NHP oocytes. This work was supported by the Tissue Distribution Program of the WaNPRC (NIH grant # R00166).